N-Trimethyl Chitosan Chloride-Coated PLGA Nanoparticles

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N-trimethyl Chitosan Chloride-Coated PLGA Nanoparticles Overcoming Multiple Barriers to Oral Insulin Absorption Jianyong Sheng, Limei Han, Jing Qin, Ge Ru, Ruixiang Li, Lihong Wu, Dongqi Cui, Pei Yang, Yuwei He, and Jianxin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b03555 • Publication Date (Web): 25 Jun 2015 Downloaded from http://pubs.acs.org on July 1, 2015

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ACS Applied Materials & Interfaces

N-trimethyl Chitosan Chloride-Coated PLGA Nanoparticles Overcoming Multiple Barriers to Oral Insulin Absorption

Jianyong Sheng a, Limei Han a, Jing Qin a, Ge Ru a, Ruixiang Li a, Lihong Wu b, Dongqi Cui b, Pei Yang a, Yuwei He a and Jianxin Wang a,*

a

Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China

b

Department of Pharmaceutics, School of Pharmacy, Heilongjiang University of Chinese Medicine, Haerbin, Heilongjiang 150040, China

* Corresponding author. Tel.: +86-21-51980088; fax: +86-21-5198 0087. E-mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract Although several strategies have been applied for oral insulin delivery to improve insulin bioavailability, little success has been achieved. To overcome multiple barriers to oral insulin absorption simultaneously, insulin-loaded N-trimethyl chitosan chloride (TMC)-coated polylactide-co-glycoside (PLGA) nanoparticles (Ins TMC-PLGA NPs) were formulated in our study. The Ins TMC-PLGA NPs were prepared using the double-emulsion solvent evaporation method and were characterized to determine their size (247.6±7.2 nm), zeta potential (45.2±4.6 mV), insulin-loading capacity (7.8±0.5%) and encapsulation efficiency (47.0±2.9%). The stability and insulin release of the nanoparticles in enzyme-containing simulated gastrointestinal fluids suggested that the TMC-PLGA NPs could partially protect insulin from enzymatic degradation. Compared with unmodified PLGA NPs, the positively charged TMC-PLGA NPs could improve the mucus penetration of insulin in mucus-secreting HT29-MTX cells, the cellular uptake of insulin via clathrin- or adsorption-mediated endocytosis in Caco-2 cells, and the permeation of insulin across a Caco-2 cell monolayer through tight junction opening. After oral administration in mice, the TMC-PLGA NPs moved more slowly through the gastrointestinal tract compared with unmodified PLGA NPs, indicating the mucoadhesive property of the nanoparticles after TMC coating. Additionally, in pharmacological studies in diabetic rats, orally administered Ins TMC-PLGA NPs produced a stronger hypoglycemic effect, with 2-fold higher relative pharmacological availability compared with unmodified NPs. In conclusion, oral insulin absorption is improved by TMC-PLGA NPs with the multiple absorption barriers overcome simultaneously. TMC-PLGA NPs may be a promising drug delivery system for oral administration of macromolecular therapeutics.

Keywords: Nanoparticles; insulin; oral delivery; N-trimethyl chitosan chloride; absorption barriers; tight junctions; mucus

2


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1. Introduction Insulin is commonly used to treat diabetes.1 Oral delivery has become the most desirable method of insulin administration because of its convenience and good patient compliance. More importantly, the physiological mechanism of insulin secretion is mimicked by oral administration.2 In particular, insulin absorbed in the intestine can go directly to the liver, the main target organ of insulin, through the portal vein, and high exposure via the peripheral circulation can be avoided.3 Consequently, oral delivery of insulin results in fewer side effects. However, the oral bioavailability of protein/peptide therapeutics such as insulin is very poor, mainly due to the following three barriers: A) Enzyme barrier: insulin is susceptible to digestion by proteases in the gastrointestinal (GI) tract.4 B) Mucus barrier: the GI tract is coated with a mucus layer that may prevent macromolecules or drug delivery systems from reaching beneath the epithelial cells.5, 6 C) Epithelial cell barrier: because of the poor cellular uptake of hydrophilic macromolecule in epithelial cells, insulin can hardly permeate the epithelial cell layer in the intestine. Meanwhile, the tight junctions (TJs) between epithelial cells prevent para-cellular transport of insulin.7 To improve the oral bioavailability of insulin, these multiple absorption barriers have to be overcome simultaneously. Previously, several strategies were used to improve oral insulin absorption.

8-13

Among these strategies, nanoparticles (NPs) may represent the most promising approach. Protein drugs encapsulated in nanoparticles are protected from enzymatic degradation in the GI tract. Moreover, the permeability of protein drugs through the mucus or epithelial cell barrier is enhanced due to the greater surface-area-to-volume ratio of nanoparticles or the modification of nanoparticles’ surface properties. 7, 13, 14 Polylactide-co-glycoside (PLGA) is approved by the US FDA and has been proven to be safe for clinical applications. PLGA nanoparticles (PLGA NPs) have gained numerous popularities in drug delivery.15, 16 Controlled release can be achieved by using PLGA NPs to effectively protect encapsulated protein drugs from enzymatic degradation in the GI tract.17-22 However, the penetration of PLGA NPs through the 4


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mucus and epithelial barriers is poo because the negative surface charge of PLGA NPs limits the nanoparticles’ interactions with the negatively charged mucus layer or cell membrane.17-20 Coating by cationic polymers through electrostatic interactions could be an effective method for modifying the surface charge of PLGA NPs. N-trimethyl chitosan chloride (TMC) is a partially quaternized derivative of chitosan and can be prepared by reductive methylation.23, 24 The positive charge, good solubility and permeation-enhancing ability of TMC are maintained in neutral-pH environments, where chitosan is insoluble and ineffective as a permeation enhancer.25, 26

As previously reported, insulin loaded TMC nanoparticles (TMC NPs) were most

commonly prepared by ionic crosslinking method.27-30 In the process of preparation, the TMC NPs were formed by self-assembly of cationic TMC and oppositely charged insulin or by addition anionic low molecular crosslinker, such as tripolyphophate (TPP) to positively charged TMC. It was reported that the insulin loaded TMC NPs possess positively charged surface, and showed good mucoadhesive and intestinal epithelium-penetrating propertie.29 However, TMC NPs disintegrate easily because the electrostatic interaction between negatively charged insulin and positively charged TMC decreases in media with an ionic strength as high as that of intestinal fluid, resulting in the fast release of insulin. In fact, up to 50% of encapsulated insulin is released from TMC NPs in PBS in just 1 h.30 With the presence of enzymes in the GI tract, this release could be even faster due to enzymatic degradation of the TMC NPs prepared by ionic crosslinking method. Although para-cellular transport of insulin is enhanced by the TJ-opening effect of TMC,29 the limited area of TJs relative to the intestinal epithelia still makes it difficult for considerable amounts of insulin to be released to effectively permeate the epithelial cell barrier. As a result, the released insulin has poor absorption efficiency and undergoes enzymatic degradation in the intestinal lumen. It was indicated that although TMC NPs prepared by ionic crosslinking method is beneficial for overcoming the mucus and epithelial cell barriers to oral insulin absorption, the ability of the nanoparticles to overcome enzyme barrier need to be improved. In our study, TMC coating of PLGA NPs was applied to combine the 5



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controlled-release

property

of

PLGA

NPs

with

the

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mucoadhesive

and

permeation-enhancing capacity of TMC. Insulin is encapsulated in the PLGA core. Meanwhile, TMC coating of the PLGA core is realized by electrostatic interactions between the negatively charged surface of PLGA core and cationic TMC. Protection of insulin in the GI tract is specifically achieved by the controlled release of insulin from our prepared TMC-coated PLGA nanoparticles (TMC-PLGA NPs) to overcome the enzyme barrier to oral insulin absorption. Moreover, it is hypothesized that the mucus barrier can be overcome by mucoadhesion of the TMC-PLGA NPs, followed by mucus penetration, and that the epithelial barrier can be overcome by improved cellular internalization of cationic TMC-PLGA NPs and enhanced para-cellular transport of insulin. By overcoming these multiple absorption barriers, oral administration of insulin-loaded TMC-PLGA NPs (Ins TMC-PLGA NPs) is expected to improve the bioavailability of insulin. The effects of TMC-PLGA NPs on overcoming the multiple barriers to oral insulin absorption were evaluated both in vitro and in vivo. The protective ability of the nanoparticles was specifically evaluated based on the stability of the nanoparticles and the release of insulin in simulated gastrointestinal fluids. In addition, mucus-producing HT29-MTX cells were applied for in vitro evaluations of the mucus-penetrating capacity of the nanoparticles, and Caco-2 cell monolayers were used to investigate the epithelia-permeating ability of the nanoparticles. Furthermore, the mucoadhesive effect of the TMC-PLGA NPs was evaluated in mice. Finally, the hypoglycemic effect and relative pharmacological availability of the nanoparticles were tested in diabetic rats. 2. Experimental methods 2.1. Materials and animals. Lakeshore® poly(lactic-co-glycolic acid) (PLGA, lactic acid:glycolic acid=50:50 and molecular weight of 20,000 Da) was kindly provided by Evonik, GmbH. (Evonik, Germany). Emprove exp® polyvinyl alcohol (PVA) 4-88 was given as a present from Merck, GmbH. (Merck, Darmstadt, Germany).Trimethyl chitosan (TMC, viscosity 10-50 mPa·s, degree of deacetylation 85%, degree of trimethyl substitution 50%) was purchased from Xingzhongcheng 6


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Chemical Co., Ltd. (Hubei, China). Porcine insulin (27 IU/g) was obtained from Wanbang Biochemical Co., Ltd. (Jiangsu, China). 125I or Rhodamine B labeled insulin was provided by Prof. Jianhua Zhu and Prof. Cong Li, respectively (School of Pharmacy, Fudan University). Fluorescent FPR648 labled PLGA (lactic acid:glycolic acid=50:50 and molelular weight of 20,000 Da) was purchased from Polyscitech (West Lafayette, IN, USA). Corning Cellgro® phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), 0.25% (w/v) trypsin-EDTA, and Dulbecco's modified essential medium (DMEM) and were all purchased from Mediatech, Inc. (VA, USA). Cell culture plates were from Corning (Corning, NY, USA). N-Acetyl-L-Cysteine (NAC) was obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). Gibco® fetal bovine serum (FBS), Alexa Fluor 488 labeled wheat germ agglutinin (WGA) and Alexa Fluor 488 labeled donkey anti-goat IgG were purchased from Life tech Inc. (Life technologies, Carlsbad, CA, USA). Occludin goat polyclonal IgG were obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). The pepsin, pancreatin, and other chemicals were purchased from Sigma (St. Louis, MO, USA). Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA, USA). HT29-MTX cell line was a kind gift from Dr. Thecla Lesuffleur (INSERM, Paris, France). Male Wistar rats weighing 200±20 g and male Kunming mice weighing 20±2 g were obtained from Sino-British SIPPR/BK Lab. Animal Co., Ltd. (Shanghai, China). All animal experiments were performed according to the Guiding Principles for the Care and Use of Experiment Animals in Fudan University (Shanghai, China). The protocols of the study were evaluated and approved by the ethical committee of Fudan University. 2.2. Preparation of NPs. Insulin loaded PLGA NPs (Ins PLGA NPs) were prepared using a double emulsion method, with slight modification from the method described previously.31 Briefly, 0.2 mL of 20 mg·mL-1 insulin PBS solution was added to 1 mL ethyl acetate containing 20 mg PLGA. A W/O primary emulsion was formed after ultrasound sonication (200 W) for 30 s in ice bath. Then 2 mL of 2% (W/V) PBS solution of polyvinyl alcohol (PVA) was added and mixed by shaking out thoroughly followed by ultrasound sonication (200 W) for 30 s in ice bath to produce a W/O/W double emulsion. The double emulsion was added dropwisely into 8 mL of 2% PBS solution of PVA and was mixed by magnetic stirring for 1 h at room 7



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temperature. The residual ethyl acetate was removed under vacuum rotary evaporation at 40°C. Aliquots of the NPs suspension were washed twice with PBS by centrifugation (15000×g, 4°C, 30 min) and resuspension. Insulin loaded TMC-PLGA NPs (Ins TMC-PLGA NPs) were prepared by the same method as above except that cationic TMC (0.8 mg) was dissolved in 2 mL of a 2% (w/v) PBS solution of PVA as the outer water phase to form double emulsion (W/O/W). 2.3. Characterization of NPs. The size and zeta potential of NPs were measured by Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The mean particle size, polydispersion index (PDI) of size, and zeta potential were recorded. All measurements were performed in triplicate. Morphological examination of PLGA NPs or TMC-PLGA NPs were performed using a transmission electron microscope (Tecnai G2 20, FEI, USA). To determine the encapsulation efficiency (EE) and drug loading capacity (DLC), the insulin-loaded nanoparticles were separated from the aqueous suspension medium by ultracentrifugation at 15,000×g and 4°C for 30 min. The amount of free insulin in the supernatant was determined using ultra-performance liquid chromatography (UPLC) (Waters Acquity UPLC H Class, Waters Corporation, USA). The UPLC system comprised a quaternary solvent manager, a TUV detector and a Acquity UPLC BEH C18 column (50×2.1 mm, 1.7 µm, 130 Å) to separate insulin. The mobile phase was a mixture of water, acetonitrile, and trifluoroacetic acid with the ratio of 68.5:31.5:0.1. The flow rate was 0.3 mL/min and the detection wavelength was set at 214 nm. Aliquot 1µL of the supernatant was injected into the UPLC system. The peak area of insulin was recorded and the concentration of free insulin in the supernatant was calculated from a standard curve. The EE and DLC was calculated using following formula: Encapsulation Efficiency (EE)(%) = Drug Loading Capacity (DLC)(%) =

total amount of insulin added-free insulin total amount insulin added total amount of insulin added - free insulin weight of nanoparticles

× 100% × 100%

2.4. Stability of NPs in simulated gastrointestinal fluids. Simulated gastric fluid without pepsin (SGFsp), simulated intestinal fluid without pancreatin (SIFsp), pepsin-containing simulated gastric fluid (SGF), and pancreatin-containing simulated intestinal fluid (SIF) were prepared according to USP35. The SGF and SIF were 35 mM NaCl, 80 mM HCl, 0.3% (w/v) pepsin, pH 1.2 and 50 mM KH2PO4, 15 mM 8


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NaOH, 1.0% (w/v) pancreatin, pH 6.8, respectively. Aliquots of Ins PLGA NPs or Ins TMC-PLGA NPs were dispersed in SGFsp, SIFsp, SGF and SIF respectively at a concentration of 0.2 mg/mL. After incubation at 37°C under agitation at 100 rpm on an orbital shaker for 2 h, nanoparticles were collected by ultracentrifugation and resuspended in water. The mean particle size and zeta potential of each sample were measured as described above and were compared with the initial data before incubation. 2.5. In-vitro drug release from NPs. The method we used was slightly modified from the previous report.20 The in vitro release profiles were studied with 0.2 mg/ml 125

I-labeled insulin (125I-Ins) loaded NPs in SGFsp, SIFsp, SGF and SIF respectively

at 37°C under agitation at 100 rpm. At predetermined time, samples were collected and centrifuged. The radioactivity of

125

I-Ins in the supernatant was counted (Wallac

Wizard 1470 Gamma counter, PerkinElmer, Waltham, USA). The release percent of 125

I-Ins from the NPs was then calculated by dividing the counts in the supernatant by

the total counts of the nanoparticles added. 2.6. Cell culture. The human colon adenocarcinoma cell lines, Caco-2 and HT29-MTX cells, were cultivated separately in 75 cm2 culturing flasks containing 10 mL Dulbecco’s Modified Medium (DMEM, Corning Cellgro, Mediatech, USA) supplemented with 10% non-essential amino acid, 10% L-glutamine, 1% penicillin (100 IU/mL) and streptomycin (100 µg/mL), and 10% fetal bovine serum. Cells were maintained in an incubator at 37°C, 95% relative humidity and 5% CO2. The culture medium was replaced with fresh one every other day until the cells reached 60-80% confluence. Cells were then detached from the culturing flask by trypsin treatment, and resuspended in fresh culture medium. For cellular uptake studies, cells were seeded onto 24-well plates (Corning, NY, USA) at a density of 5×104 cells/cm2 and cultured for 14 days. For the permeation studies on cell monolayers, Caco-2 cells were seeded onto the Millicell ® hanging cell culture insert (PET membrane, pore size 0.4 µm) (Millipore Corporation, Billerica, MA, USA). The hanging cell culture inserts were placed into 24-well plates. The cells were allowed to grow and differentiate for 21 days before use. Caco-2 cells with passage numbers between 20 and 30 and HT29-MTX cells between 14 and 20 were used. 2.7. Study of mucoadhesion and mucus-penetration using HT29-MTX cells. HT29-MTX cells were seeded onto glass-bottomed cell culture dish for CLSM visualization (Nest Biotechnology Co., Ltd, Wuxi, China) at a density of 5×104 cells/ 9



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cm2, and were cultured for 14 days. Insulin was labeled with fluorescein Rhodamine B (RB-Ins) and fluorescein FPR648 labeled PLGA (FPR648-PLGA) were used in nanoparticles preparation for in vitro visualization. Cells were rinsed with HBSS, allowed to equilibrate at 37°C for 20 min, and then incubated with 500 µL of suspension of RB-Ins loaded FPR648-PLGA NPs or TMC-FPR648-PLGA NPs in HBSS (at a concentration of 500 µg/mL of RB-Ins) at 37°C, 5% CO2 for 2 h. Subsequently, the cells were washed with ice-cold PBS and counter-stained with Alexa Fluor 488 labeled wheat germ agglutinin (WGA) (5 µg/mL) for 15 min and bis-benzimide H33342 trihydrochloride (5 µg/mL) for 20 min. After that, samples were washed with PBS and examined under confocal laser scanning microscope (CLSM, LSM710, Cal Zeiss, Jena, Germany). The images of nanoparticles or RB-Ins in the cell mucus layer or in the cell were captured. To evaluate the influence of cell mucus layer on the absorption of nanoparticles, the mucus layer of HT29-MTX cells was removed prior to the experiment, with slight modification from the method described previously.32 Briefly, HT29-MTX monolayers were washed with 10 mM N-acetyl cysteine (NAC) HBSS and incubated under agitation at 150 rpm for 1 h. Subsequently, the cells with mucus layer (without treatment of NAC, the control group) along with the cells without mucus layer (after treatment of NAC, the NAC group) were washed by HBSS for 3 times and treated with RB-Ins PLGA NP or TMC-PLGA NPs suspension (at a concentration of 500 µg/mL of RB-Ins) in HBSS at 37°C, 5% CO2 for 2 h. After that, the cells were washed to remove the unassociated nanoparticles. To measure the amount of drug that penetrated the cell mucus layer and entered the cells, the cells were treated with a 3% (v/v) formalin solution (the formalin group) to remove the mucus layer as previously reported.33 The cell lysis was accomplished in cells of all groups by ultrasound sonication (200 W, 2 s each time at a 2 s interval, 40 times) in ice bath. The amount of RB-Ins was quantified by fluorescence analysis using a microplate reader (Infinite 200 PRO microplate reader, Tecan, Switzerland) with an excitation wavelength of 566 nm and an emission wavelength of 590 nm. The protein concentration of the cell lysis were determined using a bicinichoninci acid (BCA) assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). The amount of cellular uptake of RB-Ins by HT29-MTX cells was expressed as the amount of RB-Ins associated with 1 mg of cellular protein. 2.8. Cellular uptake study using Caco-2 cells. Caco-2 cells were seeded onto 10


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glass-bottomed cell culture dish for CLSM visualization (Nest Biotechnology Co., Ltd, Wuxi, China) at a density of 5×104 cells /cm2, and were cultured for 14 days. Cells were rinsed with HBSS, allowed to equilibrate at 37℃for 20 min, and then incubated with 500 µL of a 500 µg/mL RB-Ins loaded FPR648-PLGA NPs or TMC-FPR648-PLGA NPs suspension in HBSS (at a concentration of 500 µg/mL of RB-Ins) at 37°C, 5% CO2 for 2 h. In addition, RB-Ins solution at the same concentration was set as control. After that, the cells were washed 3 times with ice-cold PBS to remove the unassociated nanoparticles. Cells were counter-stained with DAPI (4’6-diamidino-2-phenylindole) (5 µg/mL) for 10 min under light exclusion. Samples were washed with PBS and examined under CLSM (LSM710, Cal Zeiss, Jena, Germany). The intracellular CLSM images were captured. To quantitatively evaluate the cellular uptake of RB-Ins, Caco-2 cells were treated with RB-Ins PLGA NP or RB-Ins TMC-PLGA NPs suspension (at a concentration of 500 µg/mL of RB-Ins) in HBSS at 37°C, 5% CO2 for 2 h. In addition, RB-Ins solution at the same concentration was set as control. Then cells were washed to remove the unassociated nanoparticles. The cell lysis was performed and the fluorescence intensity and protein concentration of the cell lysis were determined in the same way as described above. The amount of cellular uptake of RB-Ins by Caco-2 cells was expressed as the quantity of RB-Ins associated with 1 mg of cellular protein. 2.9. Mechanism of cellular uptake in Caco-2 cells. To identify the possible cellular uptake mechanism of nanoparticles by Caco-2 cells, the cells were incubated in different conditions or treated with specific agents, with slight modification from the previously described method.29,

33

Briefly, 100 mM sodium azide, 10 µg/mL

chlorpromazine, 5 µg/mL filipin III, 10 µg/mL cytochlalasin-D or 1 mM protamine sulfate was respectively dissolved in the suspension of nanoparticles and incubated with cells for 2 h at 37°C. To test the effect of temperature on cellular uptake, cells were incubated with suspension of nanoparticles at 4°C for 2 h. In addition, the control samples were conducted at 37°C for 2 h without any treatment. The results of inhibition tests were presented as the percentage of cellular uptake amount of RB-Ins relative to the control group. 2.10. Transport through Caco-2 cell monolayers. The measurement of transepithelial electrical resistance (TEER) value and the transcelluar investigation of insulin through Caco-2 cell monolayers were carried out by the method slightly 11



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modified from the previously report.34 Prior to the transport study, the medium in the apical and basolateral chambers were replaced with pre-warmed fresh HBSS. The cells were rinsed with HBSS and allowed to equilibrate at 37°C for 20 min. Then media in the apical chamber were replaced with 0.1 mL of suspension of RB-Ins PLGA NPs or RB-Ins TMC-PLGA NPs (RB-Ins at a concentration of 500 µg/mL). At predetermined time intervals, 0.2 mL of samples were collected from the basolateral chamber. The amount of RB-Ins transported was determined as described above, and the percentage of RB-Ins transported through the cell monolayers was calculated. The apparent permeability coefficients (Papp, cm/s) of RB-Ins were calculated according to the following equation: =

!×" ×#

Where Q is the total amount of insulin permeated (ng), A is the diffusion area of the cell monolayers (cm2), c is the initial concentration of the insulin in the donor compartment (ng/cm3) and t is the total time of the experiment (s). At certain time intervals, the TEER values were measured by Millipore® Millicell-Electrical Resistance System (Millipore Corporation, MA, USA). After incubation with nanoparticles for 2 h, the cell monolayers were washed by PBS to remove the nanoparticles and subsequently cultured in fresh cell culture media. The TEER values were measured at predetermined time intervals in the next 22 h. The percentage of TEER values relative to the initial level was calculated. 2.11. Immunofluorescence imaging of occludin protein. Caco-2 cells were grown on Transwell® permeable supports (polyester membrane, pore size of 0.4 µm, Corning, NY, USA) for 21 days to confluence and treated by FPR648-PLGA NPs or TMC-FPR648-PLGA NPs suspension. Occludin protein was stained using the immunofluorescence method as reported previously.35 In brief, cells were fixed in 10% formalin, permeabilized in 1% BSA 0.4% Triton-X PBS and treated with 5% donkey serum PBS for 1 h to block nonspecific binding. The occludin goat polyclonal IgG at a concentration of 1:1000 was applied to the cells at 4°C overnight. After the washing steps, cells were treated by Alexa Fluor 488 labeled donkey anti-goat IgG (1:100 in PBS) for 1 h at room temperature. Subsequently, cells were viewed using CLSM for the tight junctions. 2.12. In vivo intestinal mucoadhesion study. Male Kunming mice weighing 20±2 g were fasted overnight, but get free to water before experiment.

125

I-Ins PLGA 12


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NPs or

125

I-Ins TMC-PLGA NPs were administrated by intragastric gavage. After

predetermined time, the mice were sacrificed by cervical dislocation. An incision was made in the abdomen. The small intestine and colon were separated. In particular, the small intestine from the upper duodenum to the lower ileum, was divided into four segments equally in length. All the intestinal segments were washed in normal saline. After that, the radioactive intensity was measured by γ-radioactive counter. The percentage of radioactive intensity of each intestinal segment to the total was calculated. 2.13. Pharmacodynamics study in diabetic rats. The method we used is slightly modified from previously reported method.36 Male Wistar rats weighing 180-220 g were fasted for 24 h, but with free access to water before intraperitoneal injection of streptozotocin (STZ) at a dose of 60 mg/kg to induce diabetes. Rats were regarded to be diabetic when their fasting blood-glucose level was higher than 15 mmol/L one week after the streptozotocin injection. The diabetic rats were fasted overnight before administrated with the following formulations: oral administration of Ins PLGA NPs (at the insulin dose of 20 IU/kg), Ins TMC-PLGA NPs (at the insulin dose of 20 IU/kg), Ins solution (50 IU/kg), PBS and subcutaneously injection of Ins solution (2 IU/kg). Before and at predetermined time intervals after dosing, one drop of blood samples were collected from the caudal vein and the blood glucose concentration was measured as previously described.36 The change in blood glucose concentration, represented by percentage to the initial value before the administration, was plotted against time. The biological activity of insulin or insulin loaded nanoparticles was calculated by dividing the blood glucose percentage by the corresponding blood glucose percentage in the control group (PBS ig. group). The area above the curve (AAC) was calculated using the trapezoidal method and was used to calculate the pharmacological availability (PA) according to the following equation:

2.14. Statistical analysis. Data were presented as the mean ± standard deviation. T-test and one-way analysis of variance (ANOVA) were used to determine significance between two groups and among more than two groups respectively. A value of P < 0.05 was considered to be significant. 13



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3. Results 3.1. Characterization of NPs. The mean particle size of the Ins TMC-PLGA NPs tested by dynamic light scattering was about 100 nm greater than that of Ins PLGA NPs (Table 1). Similar results in particle size was shown in the TEM images of PLGA NPs and TMC-PLGA NPs (Figure 1). Whereas Ins PLGA NPs had a negative zeta potential, the surface of Ins TMC-PLGA NPs was positively charged. It was indicated that the PLGA NPs were successfully coated with cationic TMC. The encapsulation efficiency (EE) and drug-loading capacity (DLC) of the TMC-PLGA NPs showed no significant differences from those of the PLGA NPs (P>0.05).

Figure 1. TEM images of PLGA NPs and TMC-PLGA NPs. Table 1: Mean size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE) and drug-loading capacity (DLC) of nanoparticles (n=3). Nanoparticles

Size (nm)

PDI

Zeta potential (mV)

EE (%)

DLC (%)

Ins PLGA NPs

132.4±5.8

0.139±0.046

-8.9±3.4

48.0±3.5

8.5±0.6

Ins TMC-PLGA NPs

247.6±7.2

0.242±0.074

45.2±4.6

47.0±2.9

7.8±0.5

3.2. Stability of NPs in simulated gastrointestinal fluids. To evaluate the stability of the nanoparticles in the GI tract, changes in the mean particle size and zeta potential of the nanoparticles after incubation in simulated gastrointestinal fluids were measured. No significant changes in the mean particle size or zeta potential of the nanoparticles were found after incubation in the enzyme-free SGFsp or SIFsp for 2 h, suggesting that most of the nanoparticles remained intact (Figure 2A). In contrast, the stability of the TMC-PLGA NPs was impaired after contact with enzymes, as indicated by a significant decrease in the zeta potential in enzyme containing SGF and SIF and a significant decrease in the mean particle size in SGF. 14


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Figure 2. (A) Size and zeta potential of PLGA NPs and TMC-PLGA NPs after 2 h incubation in SGF without pepsin (SGFsp), in SIF without pancreatin (SIFsp), in SGF with pepsin (SGF) or in SIF with pancreatin (SIF) (n=3). The size and zeta potential of the nanoparticles before incubation served as the control. Significant differences are denoted as follows: *, P

N-Trimethyl Chitosan Chloride-Coated PLGA Nanoparticles

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